Cancer gene therapy would benefit greatly from the availability of a vector that has a high efficiency of gene expression and the ability to target tumors. A number of transfection systems have been developed to deliver heterologous genes into in vivo tumors to investigate cancer gene therapy, but all have limitations. For example, retroviral vectors have been used for gene delivery because they mediate stable gene transfer with a low potential for immunogenicity; however, transfer efficiencies are relatively low (see, e.g., Di lanni et al. J. Hematother. Stem. Cell Res., 1999, 8:645-652; Morling et al., Gene Ther., 1995, 2:504-508; Lam et al., Hum. Gene Ther., 1996, 7:1415-1422; Kume et al., Stem Cells, 1999, 17:226-232) and germ line modification is a potential problem (see Thompson, Science, 1992, 257: 1854). In addition, retroviral vectors, with few exceptions, are susceptible to lysis by serum components in human blood (see Miyao et al., Hum Gene Ther, 1997, 8:1575-1583; Russell et al., Hum Gene Ther, 1995, 6:635-641 and Rother et al., J Exp Med, 1995, 182:1345-55). This greatly limits their in vivo applications. Adenoviral vectors appear to be more efficient for gene transfer in vivo, but these vectors may be used only in a localized manner, because they lack the ability to be delivered via the bloodstream (see Duncan et al., J Gen Virol., 1978, 40:45-61; Alemany et al., J Gen Virol., 2000, 81 Pt 11:2605-2609 and Alemany et al., Nat. Biotechnol., 2000, 18:723-727), and may cause toxicity to patients due to the highly immunogenic properties of adenoviral proteins (see Ginsberg, Bulletin of the New York Acad. Med., 1996, 73:53-58 and Sparer et al., J. Virol., 1997. 71:2277-2284).
Despite these advances, cancer continues to be a major public health problem requiring new solutions. Current efforts at developing therapeutic vectors founder on problems of vector safety and expression efficacy of the therapeutic gene.
Many properties of alphavirus vectors make them a desirable alternative to other virus-derived gene delivery systems being developed, including the ability to (i) rapidly engineer expression constructs, (ii) produce high-titered stocks of infectious particles, (iii) infect non-dividing cells, and (iv) attain high levels of expression (Strauss and Strauss, Microbiol. Rev. 1994, 58:491-562; Liljeström et al., Biotechnology 1991, 9:1356-1361; Bredenbeek et al., Semin. Virol. 1992, 3:297-310; Xiong et al., Science 1993, 243:1188-1191). Defective Sindbis viral vectors have been used to protect mammals from protozoan parasites, helminth parasites, ectoparasites, fungi, bacteria, and viruses (PCT Publication No. WO 94/17813).
A cDNA encoding Venezuelan Equine Encephalitis (VEE) and methods of preparing attenuated Togaviruses have been described (U.S. Pat. No. 5,185,440). Infectious Sindbis virus vectors have been prepared with heterologous sequences inserted into the structural region of the genome (U.S. Pat. No. 5,217,879). In addition, RNA vectors based on the Sindbis Defective Interfering (DI) particles with heterologous sequences have also been described (U.S. Pat. No. 5,091,309). Alphaviruses, specifically the Semliki Forest Virus, were used medically to deliver exogenous RNA encoding heterologous genes, e.g., an antigenic epitope or determinant (PCT Publications No. WO 95/27069 and WO 95/07994). Vectors for enhanced expression of heterologous sequences downstream from an alphavirus base sequence have been also disclosed (PCT Publication No. WO 95/31565). Alphavirus-based vectors were also used for protein production or expression of protein sequences for immunization (PCT Publication No. WO 92/10578). A cDNA construct for alphavirus vectors may be introduced and transcribed in animal or human cells (PCT Publication No. WO 95/27044).
Sindbis virus is a member of the alphavirus genus and has been studied extensively since its discovery in various parts of the world beginning in 1953 (see Taylor et al., Egypt. Med. Assoc., 1953, 36:489-494; Taylor et al., Am. J. Trop. Med. Hyg, 1955, 4:844-862; and Shah et al., Ind. J. Med. Res, 1960, 48:300-308). Like many other alphaviruses, Sindbis virus is transmitted to vertebrate hosts from mosquitos. Alphavirus virions consist of a nucleocapsid, wrapped inside a lipid bilayer, upon which the envelope proteins are displayed. The envelope proteins mediate binding to host cell receptors, leading to the endocytosis of the virion. Upon endocytosis, the nucleocapsid, a complex of the capsid protein and the genomic viral RNA, is deposited into the cytoplasm of the host cell. The Sindbis virus genome is a single-stranded 49S RNA of 11703 nt (Strauss et al., 1984, Virology, 133: 92-110), capped at the 5′ terminus and polyadenylated at the 3′ terminus. The genomic RNA is of (+)-sense, is infectious, and serves as mRNA in the infected cell. Translation of the genomic RNA gives rise to the nonstructural proteins, nsP1, nsP2, nsP3, and nsP4, which are produced as polyproteins and are proteolytically processed. Early during infection, the nonstructural proteins, perhaps in association with host factors, use the genomic (+)-sense RNA as template to make a full-length, complementary (−) strand RNA. The (−) strand is template for synthesis of full-length genomic RNA. An internal promoter on the (−) strand is used for transcription of a subgenomic 26S mRNA which is co-linear with the 3′ terminal one-third of the genomic RNA. This 26S subgenomic mRNA is translated to produce a structural polyprotein that undergoes co-translational and post-translational cleavages to produce the structural proteins: C (capsid), E2, and E1 (envelope). The capsid protein C encapsidates the genomic RNA to form nucleocapsids. These interact with the cytoplasmic domain of the cell surface-bound viral envelope proteins, resulting in the envelopment of the nucleocapsid inside a membrane bilayer containing the envelope proteins, and the budding of progeny virions out of the infected cell. Sindbis virus infection has been shown to induce apoptosis in a host cell (Levine et al., Nature, 1993, 361; 739-742; Jan and Griffin, J. Virol., 1999, 73:10296-10302).
Although gene transduction based on Sindbis virus has been well-studied in vitro (see Straus et al., Microbiol. Rev., 1994, 58: 491-562; Altman-Hamamdzic et al., Gene Ther., 1997, 4; 815-822; Gwag et al., Mole. Brain Research., 1998, 63: 53-61; Bredenbeek et al., J Virol, 1993, 67; 6439-6446; Liljestrom et al., Biotechnology, 1991, 9: 1356-1361; Piper et al., Meth. Cell Biol., 1994, 43:55-78; and Grusby et al., Proc Natl. Acad. Sci. USA., 1993, 90:3913-3917) and there are several reports of in vivo Sindbis virus gene transfer to the central nervous system (Duncan et al., J Gen Virol., 1978, 40:45-61; Alemany et al., J Gen Virol., 2000, 81 Pt 11:2605-2609; and Alemany et al., Nat. Biotechnol., 2000, 18:723-727) as well as to antigen presenting cells (see Tsuji et al., J Virol, 1998, 72:6907-6910; Hariharan et al., J Virol, 1998, 72:950-958; Pugachev et al., Virology, 1995, 212:587-594; and Xiong et al., Science, 1989, 243: 1188-1191), in vivo use of Sindbis virus system has been rather limited.
However, a major drawback to the use of Sindbis virus-based vectors was the fact that these vectors were thought (prior to this invention) to lack useful target cell specificity. For mammalian cells, at least one Sindbis virus receptor is a protein previously identified as the high affinity laminin receptor (HALR), whose wide distribution and highly conserved nature may be in part responsible for the broad host range of the virus (Strauss and Strauss, 1994, supra; Wang et al., J. Virol. 1992, 66:4992-5001). It was therefore thought desirable to alter the tropism of the Sindbis virus vectors to permit gene delivery specifically to certain target cell types (see PCT Publication No. WO 98/44132). Such alteration of tropism was suggested to require both the ablation of endogenous viral tropism and the introduction of novel tropism, e.g., by engineering a chimeric viral envelope protein containing an IgG binding domain of protein A.
In the mature Sindbis virus virion, a (+)-sense viral genomic RNA is complexed with capsid protein C to form icosahedral nucleocapsid that is surrounded by lipid bilayer in which two integral membrane glycoproteins, E1 and E2 are embedded (Strauss and Strauss, 1994, supra). Although E1 and E2 form a heterodimer that functions as a unit, the E2 domain appears to be particularly important for binding to cells. Monoclonal antibodies (mAbs) capable of neutralizing virus infectivity are usually E2 specific, and mutations in E2, rather than E1, are more often associated with altered host range and virulence (Stanley et al., J. Virol. 1985, 56:110-119; Olmsted et al., Virology 1986, 148:245-254; Polo et al., J. Virol. 1988, 62:2124:2133; Lustig et al., J. Virol., 1988, 62:2329-2336). Also, a Sindbis virus mutant was identified which contained an insertion in E2 and exhibited defective binding to mammalian cells (Dubuisson et al., J. Virol. 1993, 67:3363-3374).
In summary, there remains a need in the art for an effective treatment for cancer. In particular, the art needs an effective therapy that specifically targets tumor cells for destruction without significant adverse consequences for normal cells. The present invention addresses these and other needs in the art.